Project Summary/Abstract Benzodiazepines (BZDs), which enhance gamma-aminobutyric acid receptor (GABAAR) function, have many therapeutic indications, yet are one of the most abused prescription drugs. This project aims to investigate a common molecular mechanism of clinically used BZDs, and to identify peptides that act similarly to BZDs, but with specificity to the ?2?2?2 GABAAR. This specificity may allow for a compound to be largely devoid of the reinforcing properties associated with BZDs. Classical BZDs do not distinguish between different ?-subtype compositions of the ?x?x?x GABAAR. While different mechanisms of molecular action have been proposed for BZDs, it still remains unclear how their binding is coupled to an enhancement of GABAAR function. I propose that the binding of BZDs breaks existing electrostatic interactions in the GABAAR, and causes the formation of new electrostatic bonds, stabilizing the GABAAR in its positively-modified state. I will test this hypothesis using site-directed mutagenesis of the charged amino acids in the GABAAR involved in this electrostatic bond, inject Xenopus laevis oocytes with this mutated DNA, and record currents from these mutated receptors using two-electrode voltage clamp electrophysiology. Since the different ?-subtypes have a highly conserved amino acid sequence, I hypothesize that these electrostatic interactions, prior to and after BZD binding, are conserved between ? subtypes. The activity of benzodiazepines at several subtype compositions of GABAARs may underlie their reinforcing properties. Targeting only one type of GABAAR (?2?2?2) may bypass these reinforcing effects. I propose to use phage display to identify peptides that will selectively act as allosteric modulators of the ?2?2?2 GABAAR. This method of drug discovery uses filamentous bacteriophage which have sequences encoding small peptides genetically incorporated into a gene encoding a minor coat protein. A phage display library contains billions of bacteriophage with unique peptide sequences displayed on their exterior coat. I will screen these phage against HEK-293 cells expressing a non-desired target (negative selection), and then screen the phage that did not bind in the negative selection against HEK-293 cells expressing my desired target, the ?2?2?2 GABAAR (positive selection). The phage that are bound after the positive selection are phage that contain peptide sequences that can bind to the ?2?2?2 GABAAR. These peptides will be synthesized and tested electrophysiologically for activity at the ?2?2?2 GABAAR using two electrode voltage clamp electrophysiology in Xenopus laevis oocytes. Although not proposed, future experiments will test the peptides that I identify in rodents to investigate their effectiveness, and their (hopefully lack of) reinforcing properties.